Immersion liquids are used in microscopy in particular in high-resolution immersion object lenses. The optical properties of the immersion liquid are of decisive importance for a high image quality, in particular a index of refraction that is adapted to the use and a dispersion (Abbe number) that is adapted to the use, as well as a high transmission and low residual fluorescence. Furthermore, a viscosity that is suitable for handling is necessary and the immersion liquid should neither dissolve microscopy dyes nor chemically attack the samples to be examined.
Immersion object lenses, such as are used in microscopy, are calculated to the standard temperature of 23° C. (ISO 8036). Under working conditions not equal to 23° C., image errors (spherical aberrations) occur, which lead to a deterioration of the image quality. The more the temperature deviates from 23° C., the more serious the image errors are.
Temperature deviations can result from particular examination methods which make the use of tempering chambers necessary, or else are also present due to the system, e.g. through the self-heating of electronic components, for example to 27° C. or 30° C. Examination processes in which work is done at temperatures other than 23° C. are e.g. examinations at body temperature (37° C.), up to 45° C. in protein examinations under heat shock, 16-18° C. in examinations of fish, amphibians or invertebrates and 4° C. in marine biology examinations. Until now, no immersion liquids have been known which provide acceptable image qualities in these temperature ranges. In a company-internal study, image errors in immersion object lenses due to temperature influences were systematically investigated and it was shown that the greatest influence arises from the immersion liquids used.
In the case of organic liquids, the temperature coefficient of the index of refraction ne (546.1 nm) lies in the range of from approx. −3.5×104/° C. to −4.2×104/° C. This applies correspondingly to immersion liquids. In the case of optical glasses, the temperature coefficient of the index of refraction Δn/ΔT lies in the range 20-40° C. for ne at −8.1×10−6/° C. (optical glass PK51) and 10.0×10−6/° C. (optical glass SF6). If immersion liquids are used at temperatures not equal to 23° C., their viscosity-temperature behaviour must also be taken into account. Similarly to the case of mineral oil products, the viscosity increases significantly in the cold and decreases correspondingly greatly in the warm. The dependency of the viscosity on the temperature behaves exponentially. The viscosity index (according to DIN ISO 2909) is used as characteristic value for the viscosity-temperature behaviour of Newtonian fluids such as lubricating oils.
In high-resolution microscopy, in addition to the already-mentioned temperature influence, parameters such as type of object lens, working distance, cover glass thickness and index of refraction, and the index of refraction of the examined sample also have an influence on the imaging quality. As modern microscopy is evolving towards ever higher resolutions, it is necessary, to achieve high image qualities, that the immersion liquid used can be adapted to the respective preparation temperatures. In high-resolution microscopy, it can furthermore be necessary, deviating from the standard index of refraction according to ISO 8036, to prepare immersions with variable index of refraction which are set exactly to the respective examination conditions.
The reference temperature in the ISO standard for immersion liquids (ISO 8036) is 23±0.1° C., the index of refraction at 546.1 nm ne=1.5180±0.0005. The immersion liquids adapted to the standard temperature of 23° C. and available on the market do not, however, fulfil the above-described requirements.